Orcinol Assay for Hib PRP: Polyribosyl Ribitol Phosphate, is a polysaccharide of the capsule of the Hib bacterium that

exhibits activity against phagocytes. Principle: The orcinol assay measures the concentration of ribose. When heated in concentrated acid, pentoses (ribose) form furfural. This reacts with the orcinol in the presence of FeCl3 to give a green color. This reaction is restricted to free pentoses. Since ribose is used as standard and will all react, the actual amount of PRP is 2.46 times, what the assay indicates. Ribose estimation: Reagent: A. Take 50mg of ferric chloride in 100ml of Con.HCl B. Take 1g of orcinol in 10ml of ethanol. C. Mix A & B solutions Standard: Dissolve 6.25mg of ribose in 100ml of D.water (stock62.5g/ml). Take 25l, 50l, 100l, 150l, 200l and 400l into 6 tubes. Test sample: Prepare test sample containing about ~150g /ml of polysaccharide. Dilute the sample to 2.5g to 25g of ribose. Take 25l, 50l, 100l, 150l, 200l and 400l of the diluted sample in duplicate. Blank: Take a blank using 500l of d.water Procedure: Make up the volume in each tube to 500l with d.water Add 550l of the reagent Place the tubes in a boiling water-bath at 900C for 20 min and Cool in iced water. Measure the absorbance at 670nm against blank